Structure Enabled Design of BAZ2-ICR, A Chemical Probe Targeting the Bromodomains of BAZ2A and BAZ2B

The bromodomain containing proteins BAZ2A/B play essential roles in chromatin remodeling and regulation of noncoding RNAs. We present the structure based discovery of a potent, selective, and cell active inhibitor 13 (BAZ2-ICR) of the BAZ2A/B bromodomains through rapid optimization of a weakly potent starting point. A key feature of the presented inhibitors is an intramolecular aromatic stacking interaction that efficiently occupies the shallow bromodomain pockets. 13 represents an excellent chemical probe for functional studies of the BAZ2 bromodomains in vitro and in vivo.


Protein expression and purification S32
Crystallization S32 Data collection and structural determination S32

NMR methods S33
Thermal stability shift assays S33

Isothermal titration calorimetry S34
PK experiments S34 FRAP S34 Table S1. Crystallographic data collection and refinement statistics S35 Table S2. Temperature shift data S36 Figure S1. Cresset field analysis S38 Figure S2. Analysis of the torsion of the bond that connects the imidazole and the isoazole in compound 5. S38 Figure S3. 1 H-1 H NOESY spectra of 6, 7 and 9 S39 Figure S4. FRAP data S40 Figure S5. Selectivity of compound 13 and PFI-1 in cells shown by FRAP assay S41 Figure S6. PK data S42 Unless otherwise stated, reagents and solvents were purchased from commercial suppliers and used without further purification. Chromatography solvents were HPLC grade and were used without further purification. All reactions were carried out in oven-dried flasks under a positive pressure of N 2 , and air-and moisture-sensitive reagents transferred via syringe.
To a solution of the inseparable mixture from (16) (189 mg, 0.638 mmol, 1.0 equiv) in DMF (5 mL) at 0 °C was added DBDMH (102 mg, 0.357 mmol, 0.6 equiv). The mixture was allowed to warm to room temperature, and after stirring for 24 h, brine (30 mL) and EtOAc S16 (50 mL) were added. The organic layer was separated, dried (MgSO 4 ) and concentrated in vacuo. The crude material was purified by flash column chromatography, (0-20% MeOH in
The reaction mixture was then warmed up to 50 °C. After 15 hs, the reaction mixture was cooled to room temperature and partitioned between DCM (2 ml) and water (2 ml), the aqueous layer was then re-extracted with DCM (2 x 2 ml) and the combined organic layers

S32
Protein expression and purification.
BAZ2A and BAZ2B have been expressed and purified as described previously. 2

Crystallization.
All crystallization experiments were performed using sitting-drop vapour diffusion method at 4 ˚C. Initial apo crystals of BAZ2B were obtained using the protein concentration of 10-20 mg/ml in the buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM TCEP, and the crystallization reagent containing 30%-36% low molecular weight PEG smears, 0.1 M MES pH 6.0-6.5. Soaking was performed in the drop overnight using the solution prepared from the reservoir solution supplemented with 20% ethylene glycol and 6 mM X1 or X6 inhibitors.

Data collection and structural determination.
Inhibitor-soaked crystals of BAZ2B were flash-cooled in liquid nitrogen, and diffraction data were collected in-house using Rigaku FR-E Superbright. The data were processed with MOSFLM 3 and subsequently scaled using SCALA 4 from CCP4 suite. 5 Molecular replacement was performed for structure solutions using Phaser program 6 and the BAZ2B coordinates (pdb id: 4NR9) as a search model. All structures were subjected to iterative cycles of manual model building in COOT 7 alternated with refinement using REFMAC. 8 TLS definitions used in the late refinement step were calculated using TLSMD server. 9 Geometric correctness of all structures was validated with MOLPROBITY. 10 Statistics for data collection and structure refinement are summarized in Supplementary Table 1.

NMR methods.
NMR data was collected on a Bruker Avance 500 spectrometer equipped with a 5 mm BBO probe. The 1 H spectra were referenced to the internal deuterated solvent. All NMR data were acquired at the temperature of 295 K. All data were acquired using Bruker Topspin 2.1.
The 1 H-NMR spectrum was acquired using Bruker standard 1D zg30 or zg pulse sequence.
The sweep width was 20.5 ppm, and the FID contained 64k time-domain data points. The PK experiment.
The in vitro and in vivo experiments were performed as described elsewhere. 13

FRAP.
FRAP studies were performed using U2OS cells expressing a full-length BAZ2A protein chimerized with an N-terminal eGFP. Six hours after transfection 2.5 μM SAHA was added and 1 μM inhibitor was added 1 hour before imaging, which was carried out 24 hours after transfection. Full details of the FRAP experiment are as described elsewhere. 14 S35  (2) -0.6±0.1 Data represent mean of three independent measurements. * Proteins containing PHD and bromodomain. Figure S1: Cresset field analysis. Comparison of the Cresset field strengths around the two sections of compound 1 at equal field strength threshold of 2.0. Red volume indicates regions that are electron poor, and the blue volume areas that are electron rich. It can be seen that the surface of the phenyl ring shows a region that is electron poor and the triazole a region that is electron rich. This suggests that there is a favourable electronic component to the -stack formation. Figure S2: Analysis of the torsion angles that connect the isoxazole of 5 with the central imidazole. The analysis was performed using the conformational analysis tool in MOE. To generate 5, the three dimensional structure of 7 was extracted from the crystal structure (Figure 2) and the methylpyrazole replaced by the isoaxazole. The isoaxzole was allowed to relax while all other atoms were fixed in the bioactive conformation. All other parameters were used at default settings. The conformation predicted to be energetically favoured cannot bind to the protein since the methyl group is pointing in the opposite direction (left). The analysis generated one additional conformation (right). In this conformation, the methyl group points in the correct position for binding to the BAZ2 bromodomains, but the predicted energy is less favourable suggesting that is associated with a significant penalty.